Building the mitochondrial genome editing repertoire

构建线粒体基因组编辑库

• 批准号: 10447041
• 负责人: KARL J CLARK
• 金额: $ 39.75万
• 依托单位: MAYO CLINIC ROCHESTER
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-08-21 至 2024-02-27
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10447041
• 关键词: Address; Aging; Apoptosis; Binding; Biochemical Process; Cell Nucleus; Cells; DNA; DNA Binding; DNA Sequence Alteration; DNA cassette; Deoxyribonucleases; Development; Disease; Encephalopathies; Environment; Enzymes; Event; Family; Genes; Genome; Genome engineering; Genotype; Goals; Homeostasis; Human; Human Genome; Kearns-Sayre syndrome; Lactic Acidosis; Measures; Metabolism; Methods; Mitochondria; Mitochondrial DNA; Mitochondrial Diseases; Mitochondrial Encephalomyopathies; Mitochondrial Myopathies; Mitochondrial RNA; Molecular; Mutation; Myopathy; Nuclear; Nucleic Acids; Organ; Output; Pathogenicity; Pathway interactions; Patients; Physiology; Plants; Play; Point Mutation; Protein Import; Proteins; RNA; RNA Binding; RNA Editing; RNA Recognition Motif; Reagent; Reporter; Ribonucleases; Role; Somatic Cell; Stroke; Syndrome; System; Tertiary Protein Structure; Testing; Therapeutic; Transcript; Tube; Variant; Work; base; base editor; genome editing; heteroplasmy; mitochondrial genome; novel; programs; repaired; response; scaffold; somatic cell gene editing; therapeutic genome editing; tool

Abstract Mitochondria have critical normal roles in metabolism, organ homeostasis, apoptosis and aging. Mitochondria play important but still largely mysterious roles in human physiology. Mutations in both nuclear DNA associated with proteins imported into mitochondria as well as mitochondrial DNA (mtDNA) are pathogenic. Despite this clear association of genotype with disease, there are no current treatments for patients with mitochondrial disease. Mitochondria represent a unique cellular compartment with different DNA and RNA repair and editing rules. For example, DNA nucleases that introduce double strand breaks and subsequent repair in nuclear DNA induce the degradation of mtDNA. Indeed, none of the common repair pathways found in the nucleus are active in mitochondria. This proposal uses the well-established TALE-based programmable DNA binding system for targeting mtDNA and the single-tube FUSX TALE assembly system to rapidly generate any protein-based genome engineering reagents. Similarly, we use a new, protein-based and programmable RNA binding system based on PPR proteins, a class of naturally occurring, mitochondrially localized RNA editors from plants. This application harnesses the unique environment of mitochondria to generate a new toolbox to expand the repertoire of tools to edit the human genome (RFA-RM-18-017). To develop these new molecular reagents for mtDNA and mtRNA editing of somatic cells, we will conduct the following aims: I. Develop new classes of mtDNA editing tools. Enhanced approaches to the use of mitoTALENs for preferential degradation of pathogenic mtDNA variants for MELAS and KSS will be developed, including novel nuclearly encoded reporters to detect non-mitochondrial off-targeting gene editing events. A new class of TALE mitochondrial base editors will be developed to directly edit mtDNA for pathogenic variants. II. mtRNA editing tools will be generated through harnessing the PPR family of naturally occurring programmable RNA editors. We will use our new FUSR assembly system to rapidly develop optimal RNA binding reagents, including the fusion to a set of test RNA nuclease or editing protein domains. Errant fusion transcripts in mtDNA deletion or single base variants in heteroplasmic cells will be used as the test paradigm for potential RNA editing platform development with the potential use as a therapeutic. Milestones for initial stages include the establishment of novel mtDNA heteroplasmy converting mitoTALENs against MELAS and KSS followed by testing of the new mtDNA base editor. For mtRNA editing, establishing PPR scaffolding rules for mtRNA binding followed by new programmable RNA nucleases and editors will be established. Deliverables include these novel mtDNA and mtRNA editing systems as well as humans cells with matched nuclearly encoded off-target reporter cassettes for use by any mitochondrial gene editing therapeutic system.

摘要

项目成果

Mitochondrial Base Editing: Recent Advances towards Therapeutic Opportunities.

• DOI: 10.3390/ijms24065798
• 发表时间: 2023-03-18
• 期刊: International journal of molecular sciences
• 影响因子: 5.6

An optimized FusX assembly-based technique to introduce mitochondrial TC-to-TT variations in human cell lines.

• DOI: 10.1016/j.xpro.2022.101288
• 发表时间: 2022-06-17
• 期刊: STAR PROTOCOLS
• 作者: Kar, Bibekananda;Sabharwal, Ankit;Restrepo-Castillo, Santiago;Simone, Brandon W.;Clark, Karl J.;Ekker, Stephen C.
• 通讯作者: Ekker, Stephen C.